1. Field of the Invention
This invention relates to diagnostic assays for the detection of both haptens and macromolecules based on the measurement of the activity of a dehydrogenase enzyme. These assays are widely used in both clinical and non-clinical settings for the detection of analytes of interest. A significant problem in these assays is interference caused by endogenous dehydrogenases leading to elevated signals and false-positive responses. Furthermore, due to the presence of endogenous dehydrogenases in whole blood, assays based on measurement of dehydrogenase activity cannot be used with whole blood samples.
One solution that has been proposed to circumvent this problem has been to perform the assay on protein-free ultrafiltrates. However, this is cumbersome and incorporates an extra filtration step requiring special equipment into the assay procedure. It also necessitates that the test be done by trained personnel. It would be desirable to have an assay which does not require the use of specialized equipment and trained personnel. Such an assay would allow testing in a wider array of environments, more rapidly and at lower cost.
2. Summary of Related Art
False-Positive Ethanol in Clinical and Postmortem Sera by Enzymatic Assay: Elimination of Interference by Measuring Alcohol in Protein-Free Ultrafiltrate; William C. Thompson, Deepak Malhotra, David P. Schammel, Walter Blackwell, Michael E. Ward and Amitava Dasgupta; Clinical Chemistry, 40(8), 1594-5 (1994); disclosed that removing lactate dehydrogenase by ultrafiltration eliminates interference in the Emit.RTM. Ethyl Alcohol assay.
Serum-Ethanol Determination: Comparison of Lactate and Lactate Dehydrogenase Interference in Three Enzymatic Assays; Jeffrey S. Nine, M. Moraca, M. A. Virji and K. N. Rao; Journal of Analytical Toxicology, 19, 192-196 (1995); showed that elevated serum-lactate and LDH concentrations can result in varying degrees of false-positive ethyl alcohol concentrations in enzymatic assays.
Nonpolar Interactions of Inhibitors with the Nicotinamide Adenine Dinucleotide-binding Sites of L-.alpha.-Glycerophosphate Dehydrogenase; Soo Ja Kim and Bruce M. Anderson; Journal of Biological Chemistry, 244(2), 231-5 (1969); discloses the inhibition of L-.alpha.-glycerophosphate dehydrogenase by a homologous series of aliphatic carboxylic acids.
Detergents as Selective Inhibitors and Inactivators of Enzymes; M. T. Vincenzini, F. Favilli, M. Stio, P. Vanni and C. Treves; Physiological Chemistry and Physics and Medical NMR, 17, 279-295 (1985); investigated effect of detergents on pyridine-dependent dehydrogenases.
Effects of Some Antitumor Agents on Growth and Glycolytic Enzymes of the Flagellate Crithidia; Cyrus J. Bacchi, Edward I. Ciaccio and Lois E. Koren; Journal of Bacteriology, 98(1), 23-28 (1969); disclosed that agaric acid was highly inhibitory to malate and .alpha.-glycerophosphate dehydrogenase.
Catalytic Enzyme Activity Concentration in Tissues of Man, Dog, Rabbit, Guinea Pig, Rat and Mouse; J. Lindena, Ute Sommerfeld, Cornelia Hopfel and I. Trautschold; J. Clin. Chem. Clin. Biochem., 24(1), 35-47 (1986); disclosed the presence of dehydrogenase activity in postmortem tissue.
The Cytosolic and Glycosomal glyceraldehyde-3-phosphate dehydrogenase from Trypanosoma brucei: Kinetic properties and comparison with homologous enzymes; A. M. Lambeir, A. M. Loiseau, D. A. Kuntz, F. M. Vellieux, P. A. Michels and F. R. Opperdoes; Eur. J. Biochem 198(2), 429-35 (1991); studied the effects of agaric acid on the trypanosome NAD-dependent glyceraldehyde-3-phosphate dehydrogenase enzymes.
False Positive Ethanol Results by Emit.RTM.; Kathy Thedes-Reynolds and George F. Johnson; Clin. Chem., 39, 1143 (Abstract 0111) (1993); reported that the false positive ethanol result was probably due to lactate dehydrogenase catalyzed conversion of serum lactate and reagent NAD to NADH and pyruvate.